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Title:6.3 A Cryo-EM Structure of a Novel Calicivirus, Tulane Virus
Authors:Yu G, Zhang D, Guo F, Tan M, Jiang X, Jiang W
Sample:Tulane virus
Method:Single particle reconstruction (6.3 angstroms resolution)
Sample
Sample name: Tulane virus
Oligomeric state: One Tulane virus has 90 dimers forming its icosahedral capsid (T=3).
Theoretical molecular weight of the sample: 10.4
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1virusTulane virusTulane virus
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.4 mg/mL137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH 7.4Grids with sample floated on 2% uranyl acetate for 30 seconds.400 mesh copper grid with one lacy carbon layer and one layer of ultra thin carbon on top.
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE%85 KHOMEMADE PLUNGER ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD2.7 mm1347 nm4891 nm3700036475FIELD EMISSION GUNKODAK SO-163 FILM mmObjective lens astigmatism was corrected at 250,000 magnification using quadrupole stigmator.

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Liquid nitrogen cooledFEI TITAN KRIOS AUTOGRID HOLDER°° eV80 K80 K85 K0 mrad25 e/Å229-JUL-2011
Processing
Protocol:projection matching
Software:jspr.py, EMAN, EMAN2
CTF correction:each particle
Number of particles:4338
Imposed symmetry:I
Resolution by author:6.3 Å
Resolution method:FSC (at 0.143 cut-off) calculated from maps built from independently refined half datasets that were split before initial models were built, gold-standard
Processing details:4702 Tulane virus particles were selected using combined automated selection with ethan program and manual screening with boxer program in EMAN. The microscope contrast transfer function parameters for each micrograph were first determined using an automated fitting method and then manually verified/corrected using EMAN ctfit graphic program. The entire TV dataset was divided into two halves and processed independently for all the subsequent steps including construction of initial model, 2-D alignment and 3-D reconstruction. De novo initial models were constructed using the random model method in which random particle orientations were assigned and subsequently refined iteratively until convergence. The iterative refinement process including particle alignment and 3-D icosahedral reconstruction was performed using an in-house developed program jspr.py utilizing the EMAN/EMAN2 programs and library functions. The resolution was determined based on the 0.143 cutoff criterion for two truly independent reconstructions.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1906.35 μm/pixel16NIKON SUPER COOLSCAN 9000
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1IHM rigid bodyCorrelationChimeraREALProtocol: rigid body. The three chains from 1IHM were first fitted into TV density as a whole rigid body and then divided into dimers, specific chains, domains, and subdomains and fitted.